This project describes experiments designed to test the hypothesis that a mutation in the gene encoding the thiazide-sensitive Na-Cl cotransporter is the molecular basis of Gordon's syndrome (GS). Gordon's syndrome is an autosomal dominant form of hypertension associated with hyperkalemia, renal tubular acidosis, and normal renal function. Shambelan postulated that this syndrome was caused by a "chloride shunt" in the distal nephron. According to this hypothesis, the distal nephron would be transformed from a segment that reabsorbed Na in exchange for K and H to one that reabsorbed Na primarily with Cl. The predominant Na and Cl reabsorptive pathway of the mammalian distal tubule is a thiazide-sensitive Na-Cl cotransporter. Interestingly, most signs of GS are remarkably sensitive to treatment with low doses of thiazide diuretics. Thus, the thiazide- sensitive Na-Cl cotransporter is an excellent candidate gene for GS. A mutation in the thiazide-sensitive Na-Cl transporter causing GS would constitutively activate the transport protein. This transport protein was recently cloned from the bladder of the Winter Flounder and from mammals. The message for this protein is expressed in distal convoluted tubule cells and connecting tubule cells in humans. Up regulation of thiazide- sensitive Na-Cl cotransport in the connecting tubule could explain all of the clinical features of Gordon's syndrome. We have screened a cosmid library with a mammalian thiazide-sensitive transporter clone, identified four clones, and identified a polymorphic GT repeat motif within this region on chromosome 16. The aims of this project are to, l) clarify the nephron distribution of the thiazide-sensitive Na-Cl cotransporter, the bumetanide-sensitive Na-K- 2Cl cotransporter and the amiloride-sensitive Na channel in the mammalian distal nephron, 2) identify specific regulators of thiazide-sensitive Na- Cl cotransport in vitro, 3) test the hypothesis that Gordon's syndrome is linked to the thiazide-sensitive Na-Cl cotransporter gene, 4) identify the specific mutation responsible for Gordon's syndrome, and 5) express normal and mutated ion transporters in Xenopus oocytes and mammalian cell lines to screen for functional differences in transporter activity and regulatory control. The results of these studies will identify the genetic basis and the molecular physiology of an important Mendelian form of hypertension and lead to tools for both screening and treatment. The results should also provide clues to the pathogenesis of essential hypertension especially in volume dependent patients.